Hybrid Bacillus (1-3,1-4)-β-glucanases: engineering thermostable enzymes by construction of hybrid genes

Summary Hybrid (1-3,1-4)--glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)--glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)--glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)--glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65° C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70° C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)--glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.Abbreviations AMY mature form of Bacillus amyloliquefaciens (1-3,1-4)--glucanase; - MAC mature form of B. macerans (1-3,1-4)--glucanase - SUB mature form of B. subtilis (1-3,1-4)--glucanase - H(A16-M), H(A36-M), H(A78-M), H(A107-M), H(A152-M) mature forms of hybrid enzymes having 16, 36, 78, 107, 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC     

Studien zur Paläopathologie der Invertebraten III: Parasitismus bei Ammoniten

The anomalies known from ammonites (forma-types sensu Hölder) are, as far as possible, divided in possible groups of causes and reviewed with reference to possible parasitism. Previous interpretations of anomalies as caused by parasitism are discussed: forma juxtacarinata-juxtalobata, f. juxtalobata, f. verticata, f. inflata, pearls, hypertrophy; other anomalies like forma cacoptycha, f. juxtacarinata or f. chaotica may be caused only partly by parasitism. The biological evidence and plausibility of assumed parasitism is discussed. Additionally terminological suggestions for the use of “anomaly”, “Mißbildung” and the names of forma-types are made.     

Partition coefficients of plant cuticles for monoterpenes

Summary Cuticle/water partition coefficients (K_c/w) for d-limonene, -pinene and -pinene were determined by an extrapolation and a desorption method. The sorption experiments were carried out with isolated angiosperm and gymnosperm cuticles and with [¹⁴C]-labelled monoterpenes, which were obtained biosynthetically. Both methods were suitable for the determination of the K_c/w of volatile hydrophobic compounds. For the angiosperm cuticles the partition coefficients are of the order of 10⁴, which indicates a high accumulation of monoterpenes in the cuticle. The values of the conifer cuticles of Picea abies (L.) Karst. and Abies alba Mill., however, are lower due to their high lignin content. This is proved by the increase of the partition coefficients after removal of polar and phenolic components. The K_c/w can be estimated with good accuracy from the octanol/water partition coefficient, which was determined experimentally.     

Inactivation of recombinant plasmid DNA from a human erythropoietin-producing mouse cell line grown on a large scale

Summary Experiments were carried out to assess the survival of recombinant plasmid DNA during large-scale production of recombinant human erythropoietin (rhuEPO) in a fermentation pilot plant. The analyses revealed DNA-degrading activities in the fermentation broth and in the waste-water, leading to rapid destruction of plasmid DNA added to medium or waste-water. The capability of the plasmid-DNA-spiked samples to transform competent bacteria was drastically reduced. The DNA-degrading activity in the waste-waters could be blocked by addition of EDTA or by boiling, indicating the presence of DNA-degrading enzymes (DNases). No plasmid-specific DNA sequences were detected in waste-water samples by in-vitro amplification with Taqpolymerase. Genomic DNA preparations of cell debris collected from waste-water samples only contained degraded plasmid DNA. Furthermore, it was shown that intact plasmid DNA could be degraded to fragments of less than 1000 bp by incubation at 121°C for 20 min, leading to a decrease in the plasmid-specific transforming capacity by a factor of 10³ per minute. Thus, DNA from the rhuEPO production pilot plant was efficiently inactivated at three different levels: (i) in the fermentation medium (DNase), (ii) in the waste-water container (DNase), and (iii) by heat inactivation for 20 min at 120°C. These results indicate that the probability of delivery of recombinant DNA into the environment is extremely low in such biotechnological production processes. Offprint requests to: M. R. Fibi     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

Is Ap4A involved in DNA repair processes?

Hepatoma tissue culture (HTC) cells were incubated in the presence of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) to study the variations in the bisnucleosides polyphosphates (Ap4X) pool size. A transient but sensitive accumulation of these compounds is observed; if 3-aminobenzamide (3AB) which is a potent inhibitor of the ADP-ribosyltransferase (ADPRT) is added after the MNNG treatment, a more pronounced and persistent accumulation of Ap4X can be seen. A moderate heat-shock (30 min at 43 degrees C) results also in a small accumulation of Ap4X but the shape of the accumulation curve is quite different and the increase of the Ap4X pool is not sensitive to the presence of 3AB. However, both MNNG treatment and hyperthermia cause a marked inhibition of protein synthesis. On the other hand, the ADPRT activity is enhanced in the presence of MNNG whereas hyperthermia has little or a slightly inhibitory effect on this activity. These results suggest that MNNG treatment triggers an Ap4X accumulation in eukaryotic cells different from that observed after heat-shock and it seems likely that these compounds are involved in the DNA excision repair system in which the ADPRT enzyme is also implicated.     

Lack of correlation between extensive accumulation of bisnucleoside polyphosphates and the heat-shock response in eukaryotic cells

The accumulation in large amounts of bisnucleoside polyphosphates (Ap4X) after heat shock in Xenopus laevis oocytes or cultured hepatoma cells (HTC cells) is observed after exposure to temperatures of 45 degrees C or higher. The accumulation is a transient phenomenon, with the collapse in cellular ATP concentration severely affecting the rate of synthesis of Ap4X, allowing degrading activities to empty the pool of these compounds under prolonged heat shock. This accumulation of Ap4X to high levels, compared to the basic content, is only observed under conditions leading to irreversible damage, ultimately resulting in the death of the cell. It is shown that the increase in Ap4X after hyperthermia is due to the partial or almost complete inhibition of their degradation pathways, rather than to a stimulation of their rate of synthesis. Finally, the synthesis of heat-shock proteins could be observed under conditions which do not lead to important accumulation of Ap4X, therefore ruling out the possibility that these adenylylated nucleotides would behave as chemical signals ("alarmones") triggering the synthesis of heat-shock proteins. Nevertheless, on the basis of our earlier results (Guédon, G., Sovia, D., Ebel, J. P., Befort, D., and Remy, P. (1985) Embo J. 4, 3743-3749), it cannot be excluded that Ap4X might play a role in the regulation of the heat-shock response; this would, however, rely on variations in Ap4X concentrations which do not exceed a factor of 2.     

Covalent immobilization of the multienzyme enniatin synthetase

Summary The multienzyme enniatin synthetase was covalently immobilized to N-hydroxysuccinimide activated agarose. The stability of the immobilized enzyme at 25°C was enhanced compared to the soluble enzyme. Immobilization experiments also indicated that the enniatins are synthesized by a single molecule and thus do not require interactions of several enzyme molecules.     

Anaerobic degradation of 3,4,5-trimethoxybenzoate by a defined mixed culture of Acetobacterium woodii, Pelobacter acidigallici, and Desulfobacter postgatei

Abstract Acetobacterium woodii was continuously grown on 3,4,5-trimethoxybenzoate as pure culture or in commensalistic combination with Pelobacter acidigallici and Desulfobacter postgatei . Under pure culture conditions the following growth parameters were determined: μ _max= 0.112 h⁻¹, K _s= 1.07 mM, Y _max= 35 g/mol, and m = 0.22 mmol·g⁻¹·h⁻¹. In coculture with P. acidigallici the affinity for the substrate increased and the K _s value was found to be 135 μM. Under batch culture conditions mixed populations of A. woodii, P. acidigallici , and D. postgatei completely mineralized 3,4,5-trimethoxybenzoate to CO₂, whereas under continuous culture conditions more than 3 mM acetate remained unused.